Hordatine compounds and synthesis



United States Patent ABSTRACT OF THE DISCLOSURE Isolation of novel antifungal compounds and derivatives of hordatine from natural sources such as barley coleoptiles and methods of synthesis. a

This invention relates to novel hordatine derivatives and a process for the manufacture thereof. More particul-arly this invention relates to hordatine compounds of theformula wherein R represents hydrogen, acetyl or a- D-glucopy- 'ranosyl; R represents hydrogen or lower alkoxy, preferably methoxy; and n represents an integer from 1 to 4 (preferably 4). When R and R =H, the compound is designated hordatine A; when R =H and R =methoxyl the compound is designated hordatine B and Z represents a double bondor two hydrogen atoms".

The products of this invention are prepared by a method of oxidative phenol coupling utilizing the procedures of Freudenberg (Ber. 76, 997 (1943) and H. Nirnz, et al., Ber. 96, 2086 (1963)), and applying these procedures to particularly chosen substrates such as coumaroylagmatine acetate which can' be prepared from a condensation of methyl-trans-p-coumarate and agmatine monohydrochloride." 1

As the chemical structure of hordatine and its derivatives contain asymmetric carbon atoms they are therefore capable of existing in optically active forms (enantiomers) Because of the difficulty of representing thes structural differences in graphic formulae, the customary structural formulae have been used in both the specification and the claims without distinction as to the particular structural and optical configuration of the compounds. However it should be expressly understood that while no notation has been used to make the distinction referred to above, the formulae used are to be interpreted in the generic sense, that is as representing either separate isomers or the optical 'racemates. Such a formula does not merely represent the unresolved mixture of isomers. With respect to the cis-trans isomerism of the double bond in the side 3,475,459 Patented Oct. 28, 1969 tine B. Attempts to achieve a resolution of the glucosides from a natural source have been of no avail.

The compounds to which the present invention relates are useful because of their valuable antifungal properties. Thus, for example inhibition data with test organisms in the Standard Spore Drop Assay are shown below:

EXAMPLE 1 TABLE I Percent Inhibition Concentration (p.p.m.)+ 80 40 20 10 5 2.5

zbHordatineA 100 100 100 100 0 0 :EHQrda-tine A (after irradiation with 8 sunlight) 100 100 100 0 0 0 Hordat ne A, natural 100 100 100 100 46 0 Hordatrne A, by methanolysis++ 100 100 100 100 11 '0 Cournaroylagmatine 0 0 0 0 O 0 I The acetates were used. Contained some cis isomer (has. 280).

TABLE II.TEST O R GANISM: MONILINIA FR UCTICOLA Percent Inhibition of Germination Congentration, p.p.m.:

100 100 100 11 0 100 100 100 28 0 100 100 100 35 0 0 0 0 O a 0 0 0 0 O 0 TABLE III.TEST ORGANISM: MONILINIA FRUOTIC'OLA Percent Inhibition of Germination Confientration, p.p.m.:

0 o, o q 0 A=:hordatine A, diacetate, synthetic, trans isomer;

TABLE IV, [Substrate=+-'-hordatine diacetate] Percent Inhibition of, V p Germination Substrate Concentration, ppm. 40 20 10 5. 2.5

Test Organism, M0nili1tiafructic0la 100 100 100: 100 0 chain, the above comments regarding optical isomers .2

basically apply to the geometrical isomerism.

A Hordatine and its derivatives can also be isolated as one of the antifungal factors, from barley seedlings. However, the extract contains essentially the glucosides of two closely related compounds, hordatine A and horda- Test Organism, G. cingulata;

Test'Organism, Gla'merella cingulata.

TABLE V [Snbstrate: hordatine glucosides diacetates] Percent Inhibition oi Germination Substrate concentration, p.p.n' 1. 28 14 7 3. 5

Test Organism, M. fructicolai 100 100 67 0 .0 '0 '0 0 TABLE VI A [Substratez coumaroylagmatine acetate] Percent Inhibition oi Germination Substrate concentration, ppm. 80 40 20 10 Test Organism, M. fructicola 42 0 0 Test Organism, G. cingulata 0 0 0 0 TABLE VIL-TEST ORGANISM: MONILINIA FRUCTICOLA [Substratez :b-hordatlne A diacetate (synthetic)] Percent Inhibition of Germination Concentration of substrate, p.p.m 40 20 10 2.5 0

TABLE VIII.TEST O RGANISM: GLOMERELLA GIN G ULA TA Percent Inhibition of Germination Concentration of substrate, p.p.rn 40 20 5 2.5 0

TABLE IX.TEST ORGANISM: HELMIN THOSPORI UM SATIVUM Substrate concentration, p.p.m 160 80 40 20 10 6 Percent Inhibition of Germination- 100 100 100 100 0 0 EXAMPLE 2 Isolation of hardatines (natural) Unless specified otherwise the following terminology applies:

Melting points were determined on the Kofler block, optical rotations on 1% solution in water. Ultraviolet spectral constants are for solutions in 96% ethanol. Analytical and preparative thin layer chromatography (TLC) was on a microcrystalline cellulose (FMC Corporation, American Viscose Division, Newark, Del., USA.) with the upper phase of n-butanol-acetic acid-water (4:125) as irrigant and diazotized nitraniline, bromocresolgreen or the Sakaguchi reagent as indicators. Countercurrent distributions (CD) were by the single withdrawal procedure (for terminology and symbols, see E. Hecker, Verteilungsverfahren im Laboratorium, Verlag Chemie, Weinheim 1955) using 100 elements (25 ml. each phase) and the above solvent system. Distributions and other lengthy processes on dilute solutions were carried out in the dark or in orange light.

In a typical run, the basic material (5.7 g.) obtained by ion-exchange of the hot water extract of 6-day old barley coleoptiles (4.3 kg. freshweight), was fractionated by CD (300 transfers). After analysis (UV and TLC), suitable fractions were combined (Table X), evaporated to dryness in vacuo, dissolved in 96% ethanol and filtered from insolubles after several days.

Fractions B and C (hordatine M) consisted of mixture of the glucosides of transand cis-hordatines A and B.

For the constants quoted in Table XI, the fractions were precipitated as the picrates, washed with water and reconverted into the acetate form by ion-exchange.

TABLE XL-PHYSICAL CONSTANTS OF HORDATINE M l Unchanged by addition of alkali. 2 Low concentration (;i .01 mg. per spot) is required for resolution.

In another run, the picrate was reprecipitated repeatedly by slow concentration of its solution in 96% ethanol. The waxy solid (slow liquefaction above 115 C.) had A 357, 320, 2241 (6 29,400, 31,500, 48,800).

Analysis. -Calcd. for C34H48N8O9'2C6H6N3O7: C, 47.19; H, 4.65; N, 16.75; 0, 31.43; OCH 0.0. Found: C, 47.29; H, 5.17; N, 16.23; 0, 31.64; OCH 0.0.

Fraction E: Material derived from six runs was combined and refractionated according to FIG. A.

Fraction E-III-l (hordatine A, R =H and R =H and n represents 4, 99 mg. R; 54) had a +69, M 307, 298, 229m;/. (6 18,400, 18,400, 20,700) in neutral, and M 305, 297, 235m (6 18,700, 19,400, 17,000) in alkaline solution. The dipicrate, reprecipitated by careful concentration of its solution in warm methanol, was microcrystalline, M.P. l278 C., M 358, 319. 229 mp. (e 29,200, 30,200, 45,500).

Analysis. -Calcd. for C28H33N8O4'2C6H3N3O7: C, 47.62; H, 4.40; N, 19.44. Found: C, 47.79; H, 4.57; N, 19.34.

FIGURE A ,Refracticuation .of Fraction 2:

200 transfers slngle'withdrawal process E15 E14 213 E12 E11 rum-59 60-79 80-99 E18 E17 r216 -BuOH 200 transfers single withdrawal process 2: (crystals) (liquor) 2113 'mrz an].

elements No. 84-19 20-29 30-59 E1113 2.1112 mm.

450 transfers, recycling process elements No. 20-44 EIV EXAMPLE 3 Photoisomerizations An aqueous solution (1.8%) of hordatine M was irradiated in a Pyrex flask with light from an 85 W. lamp (GE CH-3). Reaction was complete after ca. 40 mins.

Rf values of the product were unchanged but the intensities of the two principal spots were reversed. Similar changes were observed for free hordatine and p-methoxycinnamoylagmatine.

EXAMPLE 4 Acetylation of hordatine M A solution of crude glucoside in acetic anhydride kept at room temperature for 3 days, was evaporated to dryness in vacuo. The product, isolated as a glass by CD (100 transfers, elements 46-52) was soluble in chloroform when freshly prepared. It has M 313,

298 my. (EZ; 176, 168) and 11 1755 (CH3COO-), 1663 (C=N),1555, 1410 Analysis..Calcd. for

CH CO, 25.8; equiv. wt. 500.5. Found: CH CO, 25.6; equiv. wt. (titration with mineral acid), 510.

EXAMPLE 5 Hydrogenation of hordatine M EXAMPLE 6 Methanolysis of hordatine M to hordatine A and hordatine B- 1 "The glucoside' (860 mg.) was refluxed gently for 1.5 hours in methanol (86 ml.) containing HCl (2% W./v.). The product was evaporated to dryness in vacuo, dissolved in water and filtered through Dowex 2-X8 (OAc'-). Filtrate and washings were concentrated to ca. 2 ml. and absorbed on Rexyn CG 51 (H+) (25 ml.).

After thorough washing with water, the Rexyn column was eluted with acetic acid (1 N in 80% methanol).

The eluates from tworuns were fractionated by CD (400 transfers) in n-butanol water. Fractions r 6155 (95 mg.) partially crystallized on evaporation. Handpicked and dried on porous tile, the crystals of hordatine A- diacetate, had M 305, 298, 229 m (6 19,800, 19,800, 22,900) (neutral and M 305, 297, 238 nm (e 22,600, 23,300, 20,300) (alkaline) and gave 100% inhibition of Monz'linia fructicolcr at 5 ppm. (standard spore drop assay, Table I). The NMR spectrum showed only a trace of absorption attributable to methoxyl.

EXAMPLE 7 Hordatine A, containing a considerable proportion of the cis isomer, was obtained by the more convenient CD (200 transfers) of a similar hydrolyzate in n-butanolwater-acetic acid. The required fractions (r 84-70), after evaporation and re-solution in water, were extracted with small volumes on n-butanol and ether (extracts discarded) and then precipitated with picric acid. After washing with water, the salt had M 359, 320, 228 mp.

Similar purifications as of Example 7, of fractions r 69-60 afforded material rich in hordatine B, with M 315, 300, 229 m (6 15,100, 15,300, 22,000). In the NMR a broad singlet at 1- 5 .81 corresponded to ca. 2H.

EXAMPLE 9 Dihydrohordatines From dihydrohordatine glucoside. The dihydroglucoside (600 mg.) was methanolyzed (2% HCl) as described above. The basic portion was fractionated by CD (300 transfers) and fractions r 84-75 were purified though the picrate and reconverted into the acetate form as described earlier. The product (22 mg.) had M 282, 228 m (a 3,750, 16,400) in neutral, M 287, 248 m (a 4,600, 14,000) in alkaline solution; a +49; the NMR spectrum did not show a detectable signal due to methoxyl.

Like work-up of fractions r 74-65 afforded a similar product (28 mg.) which exhibited methoxyl absorption (ca. 1H) in the NMR.

EXAMPLE 10 Naturally occurring hordatine A (40 mg.) was hydrogenated in acetic acid (2 ml.) over prereduced platinum oxide (14 mg, 83%). One mole-equivalent of H was absorbed. The product obtained on evaporation was identical with the above by NMR and UV spectra, but had a +44.

EXAMPLE 11 Similar hydrogenation as Example 10 of crude aglucone (84 mg.) followed by CD (700 transfers) gave dihydrohordatine (TLC, UV) containing appreciable amounts of the B component (NMR).

EXAMPLE 12 Synthesis of (i) hordatine-A A stirred solution of coumaroylagmatine acetate (200 mg.) in water ml.) at 3035 C., containing 0.002 ml. of a 1% solution of horseradish peroxidase in 2.8 M ammonium sulfate was treated dropwise during 16 hours with hydrogen peroxide solution (0.022%, 70 ml.). The solvent was removed in vacuo (40 C.) and the residue distributed by CD (200 transfers). Fractions r 85-54 contained the desired product (68.5 mg., 34 mole-percent, estimated spectrophotometrically), optically inactive but identical with hordatine A in chromatographic behaviour.

The dipicrate precipitated from water as an amorphous solid whose IR spectrum was almost identical with that of hordatine A dipicrate. On careful concentration at room temperature it separated from methanol as an oil which solidified on trituration, M.P. 12830 C. M 359, 318, 229 m (5 29,300, 31,300, 47,300).

The diacetate, recovered by ion-exchange of the dipicrate, had M 307, 298, 229 (a 19,600, 19,700, 22,100) (neutral) and M 305 ($11.), 297, 238 m (E 20,000, 20,700, 20,000) (alkaline). The NMR spectrum was identical with that of hordatine A within experimental error. Inhibition data, with Monilinia fructicola as test organism in the standard spore drop assay are given in Example 1.

EXAMPLE 13 A solution of coumaroylagmatine acetate (98 mg.) in potassium ferricyanide (0.005 M, 55 ml.) was adjusted to pH 10 with a base. After 48 hours at room tempera- EXAMPLE 14 Isolation of p-coumaroylagmatine (l-[trans-p-hydroxycinnamoylamino]-4-quanidinobutane) Countercurrent distribution of metabolic products. The basic fraction of the water-soluble products of young barley shoots was obtained by ion-exchange. The fraction (4.5-6.5 g.) was distributed by the single withdrawal procedure in n-BuOH:H O:HOAc(4:5:1)(100x 25 ml. lower phase; 300x 25 ml. upper phase). Fractions p= to =199 were collected together and evaporated to dryness in vacuo. The products of five such runs (from a total of 20 kg. of barley shoots) were combined, the aqueous solution washed with ether and the ether-insoluble material redistributed in the same solvent system (100x 25 ml. lower phase, 169x 25 ml. upper phase). Tubes p=49 to =58 contained material which gave rise to a single basic Sakaguchi-positive spot, R: 0.7 (in n-BuOH-H O-HOAc 4:5:1, Whatman No. 1 paper). Tubes =59 to =68 contained similar material (35 mg.) of

Isolation of the picrate On addition of picric acid to the above-materials in water (3 ml.), a precipitate separated which crystallized on standing. After washing with water, the salt (50 mg.) had M.P. 214, raised to 215-217" by recrystallization from aqueous methanol and aqueous ethanol. It had M 355, 310, 292 (sh.) and 223 m,

(E12,, 291, 493, 451, 512 respectively) EXAMPLE Synthesis of p-coumaroylagmatine p-Coumaroylagmatine picrate.Agmatine dihydrochloride (153 mg.) was evaporated in vacuo from 1 N sodium hydroxide solution (0.76 ml., 1 mol-equiv.). The product was heated together with methyl-trans-p-coumarate (270 mg, 2.0 mol-equiv.) at 120 C. for 24 hr., taken up in water containing a little methanol and washed with several portions of ether. The aqueous fraction, on repeated preparative thin-layer chromatography furnished the desired product as a syrup (75 mg.) which was precipitated from water as the picrate. After recrystallization from aqueous ethanol, the salt had M.P. 215-218" dec., also on admixture with the picrate of the natural compound. (Found: C, 47.9; H, 4.7; N, 19.1. C H N O required: C, 47.5; H, 4.6; N, 19.4%.) Infrared spectra (Nujol mull and solution in dimethylsulphoxide) of the two preparations were identical, as were also the U.V. spectra. The chromatographic behaviour of both products, in four different systems, was identical except that a small amount of an impurity could be detected in the synthetic material.

Conveniently agmatinemonohydrochloride is condensed with methyl-trans-p-coumarate to give coumaroylagmatine, isolated as either the acetate or the picrate. Then for example oxidative phenol coupling of coumaroylagmatine acetate leads to hordatine A.

In general the temperature and time at which the subject reactions are conducted do not appear to be critical. At the preferred reaction temperatures, highly satisfactory results can be obtained by permitting the oxidative phenol coupling to proceed for about 16 hours. In a preferred aspect of the invention a solvent such as water is used, and the solvent is preferably one in which the reaction agents are soluble therein. Other suitable solvents are lower alcohols. In an alternate synthesis it is preferred to use potassium ferricyanide and the pH adjustment is made with any suitable base. The reaction proceeds over a period of several days at ambient temperatures. However the temperature and time does not appear to be critical.

EXAMPLE 16 p-Methoxycinnamoylagmatine hydrochloride p-Methoxycinnamic acid, mg, was refluxed with thionyl chloride (1 ml.) for 10 min. The residue after evaporation was treated overnight with agmatine sulphate (230 mg.) in pyridine (2 ml.) at room temperature. The solid was filtered off, washed with a few drops of pyridine and extracted with methanol (8 ml., 5 ml.) at room temperature. Filtration of the methanol-soluble material through Dowex 2X8 (Cl) in water afforded the desired product as the hydrochloride (96 mg.). After repeated recrystallization from water, it had M.P. 212- 215 and M 304, 290, 223 mu (e 23,000, 23,900, 14,600) (Found: C, 54.8; H, 6.8; N, 16.9; C1, 11.1. C H N O Cl required: C, 55.1; H, 7.1; N, 17.2; CI, 10.9%).

EXAMPLE 17 Methylation of p-coumaroylagmatine The hydrochloride (6.5 mg.) derived by ion-exchange from metabolite picrate was refluxed for 30 min. in methanol (2 ml.) together with potassium carbonate (60 mg.) and dimethylsulphate (0.038 ml.). After dilution with water, the solution was filtered in turn through Dowex 2-X8 (OAc-) and Rexyn CG 51 (H+). The latter column was washed with water and eluted with 2 N acetic acid (75 ml.). Preparative chromatography (on Avicel, three plates, as above) of the eluate gave the desired methyl ether (located by U.V. absorption). After evaporation from a few drops of 0.1 N hydrochloric acid, it crystallized from methanol acetone. Recrystallized from water, it had M.P. 207-210, and M.P. 209-213" on admixture with the authentic p-methoxycinnamoylagmatine hydrochloride. The U.V. (unchanged by addition of OH*) and IR. (KBr disc) spectra of the two specimens were, respectively, identical.

Similar methylation of synthetic p-coumaroylagmatine gave the same compound, again characterized by M.P., mixed M.P., U.V. and LR. spectra.

I claim:

1. A compound of the formula:

NH NH (GHz)n NH o wherein R represents hydrogen, or acetyl or R represents hydrogen, or lower alkoxy; n represents an integer from 1 to 4 and Z is selected from the group consisting of a double bond or two hydrogen atoms and the diacetates thereof.

2. A compound of the formula:

NH NH (CH2); NH C NH: NH

o0 NH- (CHz)4- NH- 0 3. A compound of the formula:

O NH (CHrh NH C\ 4. The process for preparing the compound of claim 2 which comprises reacting agmatinemonohydrochloride with methyl-trans-p-cournarate and dissolving the product of the above reaction in potassium ferricyanide and compound of claim 2.

6. Dihydrohordatine diacetate. 7. Hordatine A diacetate. 8. A compound according to claim 1 which is dihydro hordatine A.

9. A compound according to claim 1 which is dihydro hordatine A.

References Cited Stoessl: Chem. Abstracts, vol. 65, p. 2195 (1966).

'ALEX MAZEL, Primary Examiner B. I. DENTZ, Assistant Examiner US. Cl. X.R.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3 ,475 ,459 October 28 1969 Albert Stoessl It is certified that error appears in the above identified patent and that said Letters Patent are hereby corrected as shown below:

Column 3, line 39, "hardatines" should read hordatines Column 10, line 19, "A" should read B Signed and sealed this 21st day of April 1970.

(SEAL) Attest:

Edward M. Fletcher, Jr. JR

Attesting Officer Commissioner of Patents 

